Metastasis is the principal cause of prostate cancer associated mortality. We have undertaken a long-term project to understand the molecules and pathways that mediate prostate cancer metastasis and to employ these molecules as tumor biomarkers as well as potential targets for therapies to treat late-stage metastatic prostate cancer. We have focused on two specific pathways that we initially found to be involved in the transition to metastatic prostate cancer. The first is thymosin ?15, a member of the ?-thymosin family. We showed that thymosin ?15 was upregulated in metastatic prostate cancer cells relative to non- or poorly metastatic cells. Although the thymosins were originally thought to act only as intracellular modulators of actin filament elongation, newer information has shown that certain ? -thymosins have extracellular activity as stimulators of angiogenesis. To date, no cell surface interactors or downstream signaling pathways have been reported to mediate the angiogenic activity of the ?-thymosins. We have found an interaction between ?-thymosins and cell surface ATP synthase, which has previously been implicated in the activity of the angiogenesis inhibitor angiostatin. We further show that ?-thymosin stimulation of vascular endothelial cell motility activates an ATP-synthase-dependent signaling pathway that operates, in part, via the P2X4 purinergic receptor. We propose to continue our studies on the role of ?-thymosins in prostate cancer with a focus on thymosin ?15, which is upregulated in prostate cancer. In our first aim, we propose the characterization of ?-thymosin-mediated signaling pathways in endothelial cells in vitro. We will: a) characterize the signaling pathways in endothelial cells that are mediated by P2X4 receptor signaling and b) identify and characterize P2X4-independent ?-thymosin signaling pathways in endothelial cells. In our second aim, we will investigate the functional role of thymosin ?15 in angiogenesis and tumor metastasis. Here, we will: a) study the effects of thymosin ?15 in in vivo angiogenesis assays;b) investigate whether modulation of thymosin ?15 influences tumor growth and metastasis in preclinical tumor model systems;and c) study whether manipulation of the purinergic receptor system modifies tumor growth and angiogenesis. Our second pathway of interest with regard to prostate cancer metastasis is mediated by a protein called antizyme and its endogenous antizyme inhibitor (AZI). Antizyme facilitates degradation of several cell cycle- related proteins and acts a tumor suppressor, whereas AZI promotes tumor cell growth. In the previous grant cycle, we showed that antizyme and AZI are both localized to centrosomes and modulate centrosome duplication. In our third aim, we will study the functional role of antizyme inhibitor in prostate cancer growth and metastasis and in centrosome duplication. We will: a) investigate whether AZI is functionally important for prostate cancer growth and metastasis;and b) characterize the role of antizyme and AZI in centriole duplication and centrosome abnormalities. PUBLIC HEALTH RELEVANCE: Metastatic prostate cancer is one of the principal causes of cancer mortality in males. We have found three proteins, thymosin beta 15, antizyme and antizyme inhibitor, which contribute to the regulation of prostate cancer metastasis. The study of these proteins can increase our understanding of the metastatic process and provide novel targets for the treatment of metastatic prostate cancer.